Cation-chloride cotransporters are typically categorized into one of three groups: Na+—K+-2 Cl− cotransporters, Na+—Cl− cotransporter, and K+—Cl− cotransporters. Potassium-chloride cotransporters (K+—Cl− cotransporters or KCCs) were first described as a potassium efflux from erythrocytes that was induced by cell swelling. The cotransport of K+ and Cl− in erythrocytes is interdependent, with a 1:1 stoichiometry and low affinity constants for both ions. KCCs belong to a gene family of electroneutral cation-chloride cotransporters; i.e., they are not influenced by membrane potential. Under most physiological conditions, they function as an efflux pathway that is involved in regulatory volume decrease. K+—Cl− cotransport is sensitive to the diuretics bumetanide and furosemide, but with much lower affinities than Na+—K+-2Cl− cotransport.
Full-length cDNAs encoding two potassium-chloride cotransporters KCC1 and KCC2 have been reported in certain mammals. While human KCC1 has been cloned (Gillen et al. (1996) J Biol Chem 271(27):16237–16244; Holtzman et al. (1998) Am J Physiol 275(4 Pt2):F550–564), KCC2 has not been cloned from in human beings. Both proteins exhibit a level of homology to other electroneutral cation-chloride cotransporters, including the bumetanide-sensitive Na+—K+-2 Cl− cotransporters NKCC1 and NKCC2 (also known as BSC2 and BSC1, respectively), and the thiazide-sensitive Na+—Cl− cotransporter NCC (also known as TSC). An alternatively-spliced form of human KCC3 has also been reported. See Hiki et.al. (1999) J Biol Chem 274: 10661–10667.
International Publication Number WO 98/29431, published Jul. 9,1998, (inventors: Lifton et al.; assignee: Yale University) discloses roles for the human thiazide-sensitive Na—Cl transporter, TSC, the human ATP-sensitive potassium channel, ROMK, and the human Na+—K+-2Cl cotransporter, NKCC2, in causing a series of abnormal or pathological conditions. Amino acid and nucleotide sequences of several human wild-type and variant TSC, NKCC2 and ROMK proteins are disclosed.
U.S. Pat. No. 4,895,807 issued Jan. 23, 1990 to Cherksey discloses a purified membrane channel protein that was found to be related to both K+ and Cl− ion transport across cellular membranes. The channel protein is described as having a molecular weight of approximately 280 to 300 kD, as determined, for example, by SDS-polyacrylamide gel electrophoresis.
International Publication Number WO 98/53067, published Nov. 28, 1998 (inventors: Bevensee et al.; assignee Yale University) discloses the isolation and purification of polypeptides and nucleic acids pertaining to sodium bicarbonate cotransporters (NBCs).
International Publication Number WO 98/37198, published Aug. 27, 1998 (inventors: Lal et al.; assignee: Incyte Pharmaceuticals Inc.) discloses a purified and isolated human sodium-dependent phosphate cotransporter (NAPTR) and purified and isolated polynucleotides encoding the same. The polypeptides and polynucleotides pertain functionally to the homeostasis of phosphate levels in the body.
International Publication Number WO 96/34288, published Oct. 31, 1996 (inventors: Ni et al.; assignee: Eli Lilly and Company, Ltd.) discloses the isolation of a sodium-dependant inorganic phosphate cotransporter and a nucleic acid enclosing the same. The cotransporter was isolated from human brain tissue. The polypeptides and polynucleotides also pertain functionally to the homeostasis of phosphate levels in the body.
U.S. Pat. No. 5,410,031 issued Apr. 25, 1995 to Wright et al. (assignee University of California System) discloses a cDNA sequence encoding an amino acid sequence corresponding to mammalian Na+/nucleoside cotransporter protein, abbreviated SNST. Thus, the encoded protein is a cotransporter that functions in conjunction with sodium ions and with nucleosides (e.g., adenosine).
U.S. Pat. No. 5,441,875 issued Aug. 15, 1995 to Hediger (assignee: Brigham and Women's Hospital) discloses the isolation and purification of a urea transporter polypeptide and to a polynucleotide encoding the same. The disclosed polypeptide thus is described as functioning in the transmembrane transport of urea.
Potassium-chloride cotransport has been described for a number of cells, tissues, and organs, including blood, skin, heart, skeletal muscle, and brain. Potassium-chloride cotransport is thought to be involved in cell volume regulation, trans-epithelial salt absorption, renal potassium secretion, and the regulation of both intra-and extra-cellular potassium and chloride ion concentrations.
Ion transporters have been implicated in a number of diseases, including hypertension, epilepsy, sickle cell anemia, Bartter's syndrome, and Meniere's disease. However, not all diseases currently thought to be associated with defective ion transport have been shown to be associated with known ion transport genes. Hence, there might be other ion transporter alleles that are defective or are affected in these diseases.
A major impediment to the study of K+—Cl− cotransport has also been the lack of specific high affinity inhibitors. Thus, further characterization of the molecular heterogeneity of potassium-chloride cotransporters has implications for the physiology and pathophysiology of a number of tissues. Additionally, the characterization of additional isoforms of KCCs would be particularly useful in screening for antibodies or pharmaceutical compositions which can modulate ion transport and hence provide treatments to ameliorate the effects of these diseases or disorders. Finally, the chromosomal localization and genomic characterization of the human KCC genes will be invaluable in the investigation of their role in monogenic disease, polygenic disease, and complex traits such as hypertension. Such characterization thus represents a long-felt and continuing need in the art.